Visualizing and Managing Multiplex Immunofluorescence Data in QuPath

In this session, we transition from theory to practice by setting up a QuPath project and mastering the art of visualizing 18-plex RareCyte Orion data. You will learn how to navigate a high-dimensional image without overwhelming your visual system. Key Topics Covered: ☑️ Visual Integrity: Why "Rendered RGB" is for PowerPoints, while "Original Pixels" or OME-TIFF is for analysis. ☑️ The Histogram "Sweet Spot": How to find the biological signal hidden among background noise. ☑️ Artifact Awareness: Recognizing data issues early in your workflow. ☑️ Scripting Basics: Using a shared script to auto-generate channel name overlays for your exports. 00:00 - Opening QuPath & Creating Your First Project Folder 01:19 - Importing Multiplex Images & Selecting Image Type 02:18 - Navigating Brightness & Contrast (Shift + C) 03:00 - The Power of Grayscale for Channel Adjustment 04:00 - Fine-tuning the Histogram: Background vs. Signal 04:53 - Identifying Saturated Pixels 06:15 - Hiding Empty Channels & Organizing Markers 07:22 - Managing Bleed-through & Autofluorescence 08:00 - Biological Surprises: Human vs. Mouse Immune Markers 09:23 - Approaches to Noisy Channels (PD-L1 & LAG-3) 11:10 - The Importance of Positive Controls 12:15 - Saving & Recalling Custom Display Settings 13:00 - Strategies for Selecting Contrasting Colors 15:45 - Troubleshooting Metadata & Spectral Errors 17:40 - Setting Up Immune Cell Visualization 18:41 - Using the Channel Viewer for Multi-marker Inspection 20:00 - Customizing the Scale Bar & UI Preferences 21:50 - Creating Fixed-size Annotations for Comparison 22:15 - Exporting Rendered RGB vs. Original Pixels 24:58 - Automation: Setting Up Your Script Directory 25:42 - Running the "Channel Names & Colors" Overlay Script 26:45 - Capturing High-quality Screenshots for Lab Meetings 28:03 - Understanding Downsampling & Whole Slide Export Resources: Step-by-Step Training Guide: Session 01 - Visualization https://saramcardle.github.io/FS2K/Session%2001-%20Visualization.html Community Support: Use the Image.sc Forum for expert help with QuPath and scripting. https://forum.image.sc/tag/qupath